The present invention relates to genetic engineering of microorganisms used in industrial fermentation processes.
An ever increasing number of products is produced by microbial fermentation at industrial scale. Such products range from primary and secondary metabolites, such as e.g. citric acid and antibiotics, respectively, to proteins, enzymes and even complete microorganisms, e.g. in the form of baker""s yeast or biomass. Traditionally, the microorganisms in question have been subjected to classical strain improvement programs which consist of successive rounds of mutagenesis and subsequent selection of mutants with improved performance. More recently, also genetic engineering, i.e. recombinant DNA technology, has been applied to industrial microorganisms. This technology has not only allowed further improvement of production levels of products naturally produced by the microorganism in question, but has also allowed the development of totally new products and/or production processes, such as e.g. the production of heterologous proteins or metabolic pathway engineering.
Genetic engineering requires the introduction and usually also the expression of a recombinant DNA molecule into the organism in question. This process is referred to as transformation. Stable transformation of a microorganism requires that the introduced recombinant DNA molecule is maintained in the cells from generation to generation, i.e. stable inheritance. There are basically two ways of maintaining the introduced recombinant DNA molecule. First, the recombinant DNA can integrate into the host cell""s genome, e.g. into one of its chromosomes. Once integrated the recombinant DNA is part of this chromosome and will thus be maintained by replication together with this chromosome. Second, the recombinant DNA can be introduced into the cell as part of a DNA molecule capable of autonomous replication, i.e. replication independent of the host""s genome. Such autonomously replicating vectors are often derived from naturally occurring plasmids or viruses which have been adapted to accommodate the incorporation of the recombinant DNA.
With respect to industrial microbial production organisms the autonomous and the integrative vector systems each have their specific advantages and disadvantages. The autonomously replicating vector systems e.g. pose restrictions on the number and/or lengths of the DNA sequences to be introduced, are generally considered to be less stable and usually provide lower expression levels per gene copy as compared to the integrative systems. Most importantly, however, for some very important industrial microorganisms, notably the filamentous fungi such as Aspergillus, Penicillium or Trichoderma, stable autonomously replicating vector systems are simply not available.
With the integrative vector systems the disadvantages depend to some extent on the approach used for integration. Integration into a predetermined genomic sequence through homologous recombination is difficult to combine with high copy numbers of the recombinant DNA molecule. Whereas random integration of multiple copies of the recombinant DNA molecule, as frequently applied in filamentous fungi, will result in unpredictable genotypes of the transformants. Not only can this lead to a loss of advantageous properties of the transformed production strain, also the unpredictable and undefined nature of such strains is less easily accepted by the registration authorities.
WO 91/00920 discloses yeast strains in which multiple copies of recombinant DNA molecules are integrated in the ribosomal DNA repeat cluster. The recombinant DNA molecules, in this case expression vectors for heterologous genes, additionally comprise both a deficient selectable marker gene as well as yeast ribosomal DNA sequences, the latter of which enable integration of the vectors in the ribosomal DNA repeat cluster through homologous recombination. The deficient selectable marker gene is required for selection and stable maintenance of strains containing multiple copies of the vectors integrated in the ribosomal DNA repeat cluster. In addition, WO 91/000920 suggests that multicopy integration in ribosomal DNA repeats might also be applied in fungi in general, including filamentous fungi such as Aspergillus species. However, the multicopy integration system disclosed in WO 91/00920 is dependent on the use of a deficient selectable marker gene. Moreover, WO 91/00920 does not provide for integration into a genomic environment which is by nature adapted to support high level RNA polymerase II transcription of protein coding genes.
Recently, ES 2 094 088 described a DNA region which is amplified in penicilin overproducing strains of penicilium chrysogenum E-1 and penicilium chrysogenum AS-P-78. The size of the amplified DNA region is described to be 75 and 106 kb, respectively, and contains the genes pcbAB, pcbC and penDE within a 16.5 kb fragment in the amplified region. It is proposed that the DNA sequences present at the left hand and right hand ends of the amplified region can be used for the construction of vectors into which marker genes have been introduced in order to promote the amplification of genetic material situated between them, in particular for obtaining strains with greater production of penicilin by random mutagenesis with nitrosoguanidine in order to increase the copy number of the vector once it has been integrated into the genome of the microorganism. However, ES 2 094 088 fails to describe whether the approach outlined above could successfully be employed. Indeed, the method described in ES 2 094 088 suffers from several drawbacks. For example, amplification of complex DNA structures occur only at low frequency. Furthermore, the use of the mutagen nitrosoguanidine results in undesired spontaneous mutations in the genome of the microorganism. Moreover, mutagenic treatment can result in the deletion of the sequences of the amplified region present in the vector and, therefore, the deletion of the gene of interest situated between them; see also Fierro, Proc. Natl. Acad. Sci. USA (1995), 6200-6204. Also, as described above the random integration of the vector into the genome of the microorganism will result in unpredictable genotypes of the transformance.
Thus, the technical problem underlying the present invention is to provide a generally applicable approach for the construction of recombinant production strains of filamentous fungi that contain multiple copies of a recombinant DNA molecule integrated in defined predetermined target loci in its genome and which system is not dependent on the use of a particular type of selectable marker for transformation.